Carbonic Anhydrase II

Aris Kaksis     Riga Stradin's University 2023

Peptides and Backbones
    Carbonic anhydrase synthesis is the Life indispensible multi functional
Attractor of organisms Self-Organisation perfect order complex reactions
composition, which as Brownian molecular engines drive the Homeostasis
for evolution and for survival. [13]
    The carbon dioxide and water protolysis functional activity reveal multiply
generated Self-Organization Attractors: pH=7.36,
synthesis of enzyme Carbonic Anhydrase reactivity,
water [H2O]=55.3 mol/Liter concentration.
    The activation from zero H2O+CO2gas
free energy content
GH2O=GCO2gas=0 kJ/mol

to protolysis products H3O++HCO3- free energy content
GH3O++GHCO3-=22.4+46.1=68.5 kJ/mol drive

the catalyze the irreversible prtolysis of carbon dioxide to bicarbonate.
    It is involved in O2aqua Hemoglobin shuttle exchange with protolysis
generate HCO3- and H+ gradients across membranes for transport down and
for osmosis of O2aqua+H2O against the gradients.
The processes connected with homeostasis respiration and photosynthesis.
CarbonicAnhydrase
    More than 100 distinct human carbonic anhydrase II (HCAII) structures are
investigated. Carbonic anhydrase, a zinc metalloenzyme, catalyzes the irreversible
protolysis of carbon dioxide to bicarbonate and hydroxonium ions.

    Multiply generated Self-Organization Attractors: pH=7.36,
synthesis of enzyme Carbonic Anhydrase reactivity,
water [H2O]=55.3 mol/Liter concentration as Brownian molecular engines
drive the irreversible Homeostasis to generate HCO3- and H+ gradients
across membranes for transport down and
for osmosis of O2aqua+H2O against the gradients.
So sustain the evolution and the survival.
That we know as respiration and photosynthesis processing.
More than 100 distinct human carbonic anhydrase (HCA) 3D structures
have been generated in last 3 decades
[LiljasA,etal.(1972)NatNewBiol235:131–137], but a structure of an HCAII in
complex with CO2 or HCO3- has remained elusive.

Contents:


I. Introduction

    The HCAII enzyme is a functional 29-kDa 260 amino acids sequence monomers shift at Thr125-Lys127 missing 126 within 259 peptide bonds consisting of a 10-stranded, twisted β-sheet . Backbone thin off

        10         20         30         40         50         60         70         80
MSHHWGYGKH NGPEHWHKDF PIAKGERQSP VDIDTHTAKY DPSLKPLSVS
YDQATSLRIL NNGHAFNVEF DDSQDKAVLK
        90        100        110        120   125  130
       140        150        160               127
GGPLDGTYRL IQFHFHWGSL DGQGSEHTVD KKKYAAELHL VHWNTKYGDF
GKAVQQPDGL AVLGIFLKVG SAKPGLQKVV
       170        180        190        200        210
        220        230        240
DVLDSIKTKG KSADFTNFDP RGLLPESLDY WTYPGSLTTP PLLECVTWIV
LKEPISVSSE QVLKFRKLNF NGEGEPEELM
       250        260
VDNWRPAQPL KNRQIKASFK    CAH2 Human                    


2VVAMarz with CO2 Carbonic anhydrase, a zinc metalloenzyme. Active sit contains zink +2 ion having coordination number 4 and coordinated with His96, His94, His119 and water Wat-318 oxygen H2O .
Four amino acids Leu198,Trp209,Val143,Val121 on the bottom and Wat-338.
The deep water Wat-338 sits in a hydrophobic pocket bottom of the active site. Wat-318 is in a hydrophilic environment toward the mouth of the active site.
    Zinc (+II) ion metalo Enzyme protein coordinative bonds with three His94, His96, His119 nitrogens and catalitic water 263 is HO- donor to O=C=O forming HCO3-.
    The active site of HCAII. The zinc ion is tetrahedrally coordinated by 3 histidines His-94, His-96, His-119 and catalytic water Wat-263.
The proton shuttle His-64, shown in both “in” is linked via Wat-292 at His96 and “out” positions Wat-318 to the catalytic water Wat-263. Hydrogen bonds are depicted as dotted lines, and waters are labeled with numbers only .
The deep water Wat-338 sits in a hydrophobic pocket bottom of the active site. Wat-318 is in a hydrophilic environment toward the mouth of the active site cone. Zn 2+ CA active site 2VVA

II. Structure of Carbonic Anhydrase II

2VVBMarz with HCO3- Carbonic anhydrase, a zinc metalloenzyme.  Active sit contains zink +2 ion having coordination number 4 and coordinated with His96, His94, His119 and water Wat-263 oxygen H2O.
Four amino acids Leu198,Trp209,Val143,Val121 on the bottom and Wat-338.
The deep water Wat-338 sits in a hydrophobic pocketottom of the active site. Wat-318 is in a hydrophilic environment toward the mouth of the active site cone .
    Zinc (+II) ion metalo Enzyme protein coordinative bonds with three His94, His96, His119 nitrogens and water 263.
    The active site of HCAII. The zinc ion is tetrahedrally coordinated by 3 histidines His-94, His-96, His-119 and catalytic water Wat-263.
The proton shuttle His-64, shown in both “in” is linked via Wat-292 at His96 and “out” positions Wat-318 to the catalytic water Wat-263. Hydrogen bonds are depicted as dotted lines, and waters are labeled with numbers only .
The deep water Wat-338 sits in a hydrophobic pocketottom of the active site. Wat-318 is in a hydrophilic environment toward the mouth of the active site cone .
Zn 2+ CA active site Left 2VVA left at first
Zn 2+ CA active site 2VVB


IV. CO2 + 2 H2O<=>H3O++ HCO3- mehanism.

III. Structure of Carbonic Anhydrase II

2CBAMarz Carbonic anhydrase, a zinc metalloenzyme.  Zinc (+II) ion metalo Enzyme protein coordinative bonds with three His94, His96, His119 nitrogens and water 263.
    Active sit contains zink +2 ion having coordination number 4 and coordinated with His96, His94, His119 and water Wat263 oxygen H2O and surrounding water Wat292,Wat318,Wat338.
    The active site of HCAII. The zinc ion is tetrahedrally coordinated by 3 histidines His-94, His-96, His-119 and catalytic water Wat-263.
The proton shuttle His-64, shown in both “in” is linked via Wat-292 at His96 and “out” positions Wat-318 to the catalytic water Wat-263. Hydrogen bonds are depicted as dotted lines, and waters are labeled with numbers only .
The deep water Wat-338 sits in a hydrophobic pocketottom of the active site. Wat-318 is in a hydrophilic environment toward the mouth of the active site cone. Zn 2+ CA active site 2CBA.
Carbonic Anhydrase II 2CBA Reaction.
The HCAII enzyme is a functional 29-kDa monomer consisting of a 10-stranded, twisted β-sheet .
2VVAMarz with CO2 Carbonic anhydrase, a zinc metalloenzyme.  Active sit contains zink +2 ion having coordination number 4 and coordinated with His96, His94, His119 and water Wat-263 oxygen H2O .
    Zinc (+II) ion metalo Enzyme protein coordinative bonds with three His94, His96, His119 nitrogens and water 263.
     The active site of HCAII. The zinc ion is tetrahedrally coordinated by 3 histidines His-94, His-96, His-119 and catalytic water Wat-263.
The proton shuttle His-64, shown in both “in” is linked via Wat-292 at His96 and “out” positions Wat-318 to the catalytic water Wat-263. Hydrogen bonds are depicted as dotted lines, and waters are labeled with numbers only .
    The deep water Wat-338 sits in a hydrophobic pocketottom of the active site. Wat-318 is in a hydrophilic environment toward the mouth of the active site cone.
Zn 2+ CA and CO2 Wat-263 active site 2VVA

II. Structure of Carbonic Anhydrase II with HCO3-

2VVBMarz Carbonic anhydrase, a zinc metalloenzyme. Active sit contains zink +2 ion having coordination number 4 and coordinated with His96, His94, His119 and water Wat-263 oxygen H2 O .
III. Zinc (+II) ion metalo Enzyme protein coordinative bonds with three His94, His96, His119 nitrogens and water 263.

    The active site of HCAII. The zinc ion is tetrahedrally coordinated by 3 histidines His-94, His-96, His-119 and catalytic water Wat-263.
The proton shuttle His-64, shown in both “in” is linked via Wat-292 at His96 and “out” positions Wat-318 to the catalytic water Wat-263.     Hydrogen bonds are depicted as dotted lines, and waters are labeled with numbers only .
The deep water Wat-338 sits in a hydrophobic pocketottom of the active site. Wat-318 is in a hydrophilic environment toward the mouth of the active site cone.
Zn 2+ CA and CO2 Wat-263 active site 2VVA left at first.
Zn 2+ CA and OHCO2- active site 2VVB

I. Neutron diffraction of acetazolamide(AZM)-bound human CA2 inhibitor

    The HCAII enzyme inhibitor shift at Thr125-Lys127 missing 126 within 259 peptide bonds consisting of a 10-stranded, twisted β-sheet . Backbone thin off
4G0CMarz with CO2 Carbonic anhydrase, a zinc metalloenzyme. Active sit contains zink +2 ion having coordination number 4 and coordinated with His96, His94, His119 and AZM nitrogen AZM-N distance 2.381 Å.
Four amino acids Leu198,Trp209,Val143,Val121 on the bottom and Wat-338.
The deep water Wat-338 sits in a hydrophobic pocket bottom of the active site. Wat-318 is in a hydrophilic environment toward the mouth of the active site.
    Zinc (+II) ion metalo Enzyme protein coordinative bonds with three His94, His96, His119 nitrogens and catalitic water 263 replaced AZM-N salt brige .
    The active site of HCAII. The zinc ion is tetrahedrally coordinated by 3 histidines His-94, His-96, His-119 and AZM-N salt bridge as more stabile internal complex.
The proton shuttle His-64, shown in one “out” positions. Remove and havy water DOD .
The deep water Wat-338 sits in a hydrophobic pocket bottom of the active site. Wat-318 is in a hydrophilic environment toward the mouth of the active site cone. Zn 2+ CA active site 4G0C

 


V. Synthesis of Carbonic Anhydrase CA indispensable Attractor.

http://aris.gusc.lv/ChemFiles/CA/CApH7-36.pdf
    Carbonic Anhydrase reactivity and generate Physiologic buffer solutions total
equilibrium value pH=7.36 as Self-Organization Attractors. In reaction :
CO2+2H2O products (CO2aqua) HCO3-+H3O++Q
accumulate free energy content G(HCO3-+H3O+)=8.38 kJ/mol+60 kJ/mol is
indispensable for functional activity of bicarbonate buffer system on the planet
Earth for perfect reactions order in homeostasis complex processes.
    CO2 no reaction with water H2O at absence of CA. CO2 is small soluble and slow
react with OH-. Solubility CO2gas +H2O+ΔG<=>CO2aqua+Q product constant:
KspCO2aqua)a=[CO2aqua]/[CO2gas]/[H2O]=0.034 is unfavored but exothermic
ΔHHess=ΔHCO2aq-ΔHCO2gas=-20.3 kJ/mol.

ΔGspCO2aqua=-R•T•ln(KspCO2aqua)=-8.3144*298.15*ln(0.03397)/1000=

=-8.3144*298.15*ln(0.03397)/1000=8.385 kJ/mol minimum.
    Air 0.04 % mol fraction [CO2gas]=0.0004 dissolute concentration is:
[CO2aqua]=KspCO2aqua*[H2O]*[CO2gas]=0.034*55.35*0.0004=0.0007512 M.
Carbon dioxide CO2 aqua react with OH- times 1016.5 slower about neutralization
reaction: H3O++HCO3-=>CO2aqua+2H2O+ΔG+Q,
because neutralization velocity constant is k2=5.17*1018 M−2s−1,
but in reaction with OH- ions: CO2aqua+OH-=>HCO3- velocity constant is
k1OH=1.5*102 M−2s−1, however reaction is exothermic with heat production Q:
ΔHHess=ΔH°HCO3-ΔH°CO2-ΔH°OH=-48.68 kJ/mol.
  CA Carbonic Anhydrase protolytic reactivity create functional active bicarbonate buffer
Self-Organization Attractor pH=7.36 with generate concentration gradients for transport
H3O
+, HCO3-, CO2aqua down and osmosis against concentration gradient. [9]
   CA Carbonic Anhydrase high rate protolysis reaction of CO2aqua with two water molecules: CO2aqua+2H2O+ΔG+Q=v1CA>H3O++HCO3- and
velocity constant is: k1CO2aqua=1.5×108 M−2s−1. [9]

Neutralization H3O++HCO3-<=CA>CO2aqua+2H2O
velocity constant is times 1010.54
higher about Carbonic Anhydrase velocity constant:
k2/k1CO2aaqua=5.17*1018/1.5/108=1010.54.

CA protolysis equilibrium constant have calculated in the velocity constants ratio
expression:
KeqCAHCO3aqua=k1CO2aqua/k2=[H3O+]*[HCO3-]/[CO2aqua]/[H2O]2=

=Ka_CO2aqua/[H2O]2=10-7.0512/55.32=2.906*10-11.
Bicarbonate buffer system acid protolysis constant pKa_CO2aqua=7.0512
is friendly to pH=7.36:
Original pKa_CO2aqua=7.0512 obtained and calculate for BUFFER solution. [1]

High rate protolysis Attractor pH=7.36 according Henderson-Haselbah equation
with equilibrium constant value pKa=7.0512, which is frendly to Attractor 7.36:
pH=7.0512 +log([HCO3-]/[CO2aqua])=7.36


HCO3-0%                         50% salt – buffer system base 100%
CO2+ 2H2O100%            50% weak acid buffer component 0%
    Buffer region middle point over inflection point :
pH=pK=7.0512
as ratio [HCO3-]/[CO2aqua]=1 is one or
buffer component concentrations are equal
as well as bicarbonate concentration is
equal to dissolved in blood.  
At 7.36 = pH ratio is [HCO3-]/[CO2aqua]=2.036/1
    As soon as H+ concentration grows for some reason,
    Carbonic anhydrase equilibrium is shifted to left and
CO2 transported out together as  H+ and HCO3- ions by respiration in lungs and
acid concentration decreases. If acid concentration decreases,
Carbonic anhydrase equilibrium is shifted to the right and
the extra amount of HCO3- through kidneys passes
into urine and is transported out.
    Bicarbonate channels in kidney cells are open at high values of pH>7.36 from side
of blood circulation, but lungs channel transport down concentration gradients due to
Hemoglobine shuttle exchange releases H+ and HCO3- ions due to adsorption of
oxygen O2aqua. [6] .
    The alkaline reserve 2.036/1=[HCO3-]/[CO2] of the organism
can be controlled by adding H2SO4 to a sample of blood (H2SO4
reacts with HCO3- and the CO2 is liberated).
    If 56.23 mL of gaseous CO2 are liberated from 100 mL of blood,
the alkaline reserve is normal and total alkaline reserve
amount concentration 0.023M = [HCO3-]+[CO2] is normal
as [HCO3-] = 0.0154 M, [CO2]=0.0076M.

VI. CA high rate protolysis attracors for HOMEOSTASIS

Carbonic anhydrase change the conditions for equilibrium :
CO2aqua+2H2O+ΔGeqCAHCO3aqua+Q<=CA=>H3O++ HCO3-;
KeqCAHCO3aqua=k1CO2aqua/k2=[H3O+]*[HCO3-]/[CO2aqua]/[H2O]2=

=Ka_CO2aqua/[H2O]2=10-7.0512/55.32=2.906*10-11=

KeqCAHCO3aqua= 1/34412000000=10-10.54.

Free energy change:
ΔGeqCAHCO3aqua=-R•T•ln(KeqCAHCO3aqua)=

=-8.3144•298.15•ln(1/34412000000)=60.14 kJ/mol.
thermodynamically from :
ΔGeqCAHCO3aqua= 60.14 kJ/mol

Neutralization H3O++ HCO3-<=CA>CO2aqua+2H2O velocity constant
is times 1010.54 higher about Carbonic Anhydrase velocity constant:
k2/k1CO2aqua=5.16885*10^18/1.5/10^8=1010.54.

Reaction CO2aqua+OH-=>HCO3- is favored
ΔGHessHCO3-aqua=GHCO3-aqua-(GCO2aqua+GOH-)=

=46,08-(8,379+77,36)=-39,66 kJ/mol
ΔGeqOH= 39.66 kJ/mol

with equilibrium constant:
[HCO3-]/[CO2aqua]/[OH-]=KeqHCO3-=EXP(-ΔGeqHCO3-/R/T)=

=EXP(39659/8.3144/298.15)=8871734=10^6.948,
with exothermic heat production Q:
ΔHHess=ΔH°HCO3--ΔH°CO2aqua-ΔH°OH-=-48.68 kJ/mol.
HCO3-=>CO2aqua+OH- :
k1OH-/k2HCO3-=KeqHCO3-=1.5*10^2/k2HCO3-=8871734;

k2HCO3-=k1OH-/KeqHCO3-=1.5*10^2/8871734=1.6908*10^-5.
Reaction with OH- ions is millions to hundred millons 10^6-10^8 times
slower k1OH-=1.5*10^2 to k1OH-=1.5 about
CA Carbonic Anhydrase velocity constant k1CO2aqua=1.5×108 M−2s−1. [9]
with heat production Q:

ΔHHessHCO3-aqua=ΔH°HCO3-aqua-(ΔH°CO2aqua+ΔH°OH-)==-48.68 kJ/mol.

Via change the equilibrium constant from KeqCAHCO3aqua= 10-10.54

to KaHCO3aqua= 10-7.0512 or its exponennce pKaHCO3aqua= 7,0512 constant
to classic acid constant:

KaHCO3aqua=KeqCAHCO3aqua*[H2O]2=10^(-7.0512)=10^-pKaHCO3aqua.

very close to pH value of blood 7.36.

VI. Concentration gradiets for transport down and for osmosis against concentration gradients


    Membrane penetrating reaction of H3O+and HCO3- is
driven by exoergic conditions of free energy change
ΔG>0 negative ΔG=-60.145 kJ/mol through proton H+ and
bicarbonate HCO3- channel crossing cell membrane.
Membrane is equipped by aquaporins, which are
water and solute oxygen O=O permeable in both directions
crossing membrane. For protons crossing the membrane
through proton channels, necessary water molecules
locate both side of the membrane and aquaporins are supplier
of water H2O molecules.

Inside of cell cytosolAqon alveolar surface in lungs
as moisture H2O is present both side of membrane behalf of aquaporins.
Membrane penetrating reaction of H+and HCO3- is
driven by exoergic conditions of free energy change
ΔG>0 negative ΔG=-60 kJ/mol through proton H+ and
bicarbonate HCO3- channel crossing cell membrane.

Inside of cell cytosolChannelalveolar surface in lungs .

References
1.Pharmacol Ther. 1997;74(1):1-20. 1CA2
2.Proteins. 1993 Sep;17(1):93-106. 1CAM
3.J Mol Biol. 1992 Oct 20;227(4):1192-204.Abstract 2CBA
4.J Med Chem. 1995 Jun 23;38(13):2286-91.Abstract 1CNW
5.Martin DP, Hann ZS, Cohen SM. University of California, San Diego , United States. Abstract 4JSW
6.Martin DP, Hann ZS, Cohen SM. University of California, San Diego , United States. Abstract 4JSS
7.PNAS June 30, 2009 vol. 106 no. 26 10609-10613 2VVA=CO2, 2VVB=HCO3- CARBON ANHYDRASE 2CBA
8.January 2004 Molecule of the Month by Shuchismita Dutta and David Goodsell
1CA2 1CAM 1DDZ 1THJ inh 1CNW
9.University Alberta Data Tables Molar Thermodinamic Properties of Pure Substances,
http://www.vhem.ualberta.ca/
10.4.A.M.Suchotina Handbook of ElectroChemistry Petersborg 1981."Chimia"
11.Daniel C.Harris, "Quantitative chemical analysis". W.H.Freeman and Company , 5th ed.1999, p545.
12.J Am Chem Soc.2012 Sep 12;134(36):1472614729. 5G0C
13. Kaksis A. The Biosphere Self-Organization Attractors drive perfect order homeostasis reactions to link bioenergetic with functionally activate oxygen and carbon dioxide molecules. 7th International Conference on New Trends in Chemistry September 25-26, 2021.27-32.
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